The article describes the method for estimating pepsin, trypsin, papain, and cathepsin using hemoglobin as a substrate. Hemoglobin is a reproducible substrate that is digested under standard conditions, and the amount of unprecipitated protein split products is measured using a phenol reagent that reacts with tyrosine and tryptophan to produce a blue color. The method has been refined over time, with improvements in preparation, standardization, and procedures. The hemoglobin substrates for pepsin, trypsin, and papain are the same as originally described, while the cathepsin substrate now includes 0.001 M ammonium sulfate to increase digestion rate. The preparation of hemoglobin involves centrifugation, dialysis, and storage at low temperatures. The phenol reagent is diluted and used to measure the color of split products. The estimation procedures for each enzyme are similar, with variations in digestion temperature and time. The activity units are defined based on the amount of split products produced, and curves are used to relate activity units to color values. The methods have been standardized, and the curves for activity units are provided for each enzyme. The article also discusses the preparation of hemoglobin substrates, the digestion process, and the calculation of activity units. The methods are applicable to other proteinases, but a new curve must be constructed for each. The activity units are defined as the amount of enzyme that produces a specific color value in the digestion products. The standard temperatures for the enzymes are 25°C for pepsin and trypsin, 37°C for cathepsin, and 25°C for papain. The methods have been refined and standardized, and the procedures are described in detail.The article describes the method for estimating pepsin, trypsin, papain, and cathepsin using hemoglobin as a substrate. Hemoglobin is a reproducible substrate that is digested under standard conditions, and the amount of unprecipitated protein split products is measured using a phenol reagent that reacts with tyrosine and tryptophan to produce a blue color. The method has been refined over time, with improvements in preparation, standardization, and procedures. The hemoglobin substrates for pepsin, trypsin, and papain are the same as originally described, while the cathepsin substrate now includes 0.001 M ammonium sulfate to increase digestion rate. The preparation of hemoglobin involves centrifugation, dialysis, and storage at low temperatures. The phenol reagent is diluted and used to measure the color of split products. The estimation procedures for each enzyme are similar, with variations in digestion temperature and time. The activity units are defined based on the amount of split products produced, and curves are used to relate activity units to color values. The methods have been standardized, and the curves for activity units are provided for each enzyme. The article also discusses the preparation of hemoglobin substrates, the digestion process, and the calculation of activity units. The methods are applicable to other proteinases, but a new curve must be constructed for each. The activity units are defined as the amount of enzyme that produces a specific color value in the digestion products. The standard temperatures for the enzymes are 25°C for pepsin and trypsin, 37°C for cathepsin, and 25°C for papain. The methods have been refined and standardized, and the procedures are described in detail.